Evaluation of lipid-based nanoparticles as vectors for visual optogenetic therapy
- Autores: José David Celdrán
- Directores de la Tesis: Eduardo Fernández Jover (dir. tes.), Lawrence Humphreys (codir. tes.)
- Lectura: En la Universidad Miguel Hernández de Elche ( España ) en 2025
- Idioma: español
- Tribunal Calificador de la Tesis: Joaquín Rueda Puente (presid.), Marcelino Aviles Trigueros (secret.), Pedro de la Villa Polo (voc.)
- Programa de doctorado: Programa de Doctorado en Bioingeniería por la Universidad Miguel Hernández de Elche
- Materias:
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- Resumen
Blindness and visual impairment affect millions of people around the world. In developed countries, most of these types of blindness can be prevented through a healthy lifestyle, however, they can be hard to treat if they are in the late stages of the disease. These include glaucoma, age-macular degeneration, and diabetic retinopathy. However, there is a subset of diseases called inherited retinal dystrophies, which are hereditary diseases that degenerate the retina and cannot be prevented nor treated properly currently.
Retina dystrophies have been treated using different approaches. Gene therapy has been employed to deliver functional copies of vision-related genes in retinas. Other therapies have used electric neuroprostheses to stimulate remaining retinal cells. However, gene therapy is difficult to universalize due to the genetic diversity of these diseases, and electric neuroprostheses lack cell specificity.
In this regard, optogenetics, a method that introduces genes that codify for photosensitive proteins and allows precise control of the cells using light, is a promising therapy without the problems of gene therapy and electric neuroprostheses. Moreover, optogenetic neuroprostheses could activate visual cortex using light in blind people, potentially treating any blindness with more specificity than electric neuroprostheses. To carry optogenetic genes into cells, viral vectors are commonly used, but they present problems such as immunogenicity and restrictions in the size of the genetic cargo they can carry. In this situation, non-viral lipid-based vectors could deliver optogenetic genes in cells without the disadvantages of viral vectors.
On this basis, the purpose of this thesis is to evaluate the use of lipid-based vectors for optogenetic delivery in central nervous system (CNS) neurons, testing whether the cells transfected with these vectors and express the optogenetic proteins are morphologically and electrophysiologically healthy, as well as these vectors affect cell viability, checking whether these vectors could be used in future visual restoration therapies based in optogenetics.
As a preliminary study, a serotype 9 adeno-associated (AAV9) viral vector and electroporation (a physical non-viral vector) were used to deliver a gene coding for the optogenetic protein ChrimsonR in the retinas of rd10 mice, a well-known animal model for retinal degeneration. After immunohistochemistry, visual task, and electrophysiological analysis, results showed that retinas transduced with AAV9 showed expression of ChrimsonR in retinal ganglion cells (RGCs), some visual behavior in the visual task, and precise control of cells from ganglion cell layer (GCL) activity using light. On the other hand, electroporated retinas had expression of ChrimsonR in RGCs, but not visual behaviour nor electrophysiological activity. Even with these inconveniences, electroporation served as a positive control for non-viral delivery of ChrimsonR.
In the second study and publication, "Assessment of Different Niosome Formulations for Optogenetic Applications: Morphological and Electrophysiological Effects", the capacity of different niosome formulations to deliver optogenetic genes into cortical neurons in vitro was evaluated. Although niosomes delivered effectively the optogenetic genes into cortical neurons, morphological, electrophysiological, and cell viability results showed that neurons had reduced dendritic arbors, reduced electrophysiolo gicalresponses, and a decline in cell viability compared to controls, indicating that, although the optogenetic gene delivery was effective, there is room for improvement.
In the third study, lipid nanoparticles (LNPs) were used for optogenetic delivery of the ChrimsonR protein in cortical neurons in vitro, as well as in rd10 mice retina. Results showed that LNPs not only effectively delivered the ChrimsonR gene into cortical neurons, but also had no reduction in several dendritic parameters and transfected more neurons than controls, however, electrophysiology was reduced compared to controls, indicating there is still room for improvement. Regarding retinal delivery, LNPs effectively could deliver the ChrimsonR gene in RGCs in rd10 mice retina, showing promise for optogenetic delivery mediated by a non-viral vector in the retina.
In conclusion, lipid-based vectors, such as niosomes and LNPs, can effectively deliver optogenetic genes in neurons, and here, to the best of our knowledge, we are the first lab in the world to accomplishing this. However, these vectors affect significantly some fundamental parameters of neurons, highlighting the need to better understand how they work at the molecular level, to improve on these promising results.
